Lactoferrin-based food supplements trigger toxin production of enteropathogenic Bacillus cereus

Lactoferrin is an iron-binding glycoprotein exhibiting antibacterial, antiviral, antifungal, antiparasitic, antiinflammatory, antianaemic and anticarcinogenic properties. While its inhibitory effects against bacterial pathogens are well investigated, little is known about its influence on the production and/or mode of action of bacterial toxins. Thus, the present study aimed to determine the impact of food supplements based on bovine lactoferrin on Bacillus cereus enterotoxin production. First, strain-specific growth inhibition of three representative isolates was observed in minimal medium with 1 or 10 mg/mL of a lactoferrin-based food supplement, designated as product no. 1. Growth inhibition did not result from iron deficiency. In contrast to that, all three strains showed increased amounts of enterotoxin component NheB in the supernatant, which corresponded with cytotoxicity. Moreover, lactoferrin product no. 1 enhanced NheB production of further 20 out of 28 B. cereus and Bacillus thuringiensis strains. These findings again suggested a strain-specific response toward lactoferrin. Product-specific differences also became apparent comparing the influence of further six products on highly responsive strain INRA C3. Highest toxin titres were detected after exposure to products no. 7, 1 and 2, containing no ingredients except pure bovine lactoferrin. INRA C3 was also used to determine the transcriptional response toward lactoferrin exposure via RNA sequencing. As control, iron-free medium was also included, which resulted in down-regulation of eight genes, mainly involved in amino acid metabolism, and in up-regulation of 52 genes, mainly involved in iron transport, uptake and utilization. In contrast to that, 153 genes were down-regulated in the presence of lactoferrin, including genes involved in flagellar assembly, motility, chemotaxis and sporulation as well as genes encoding regulatory proteins, transporters, heat and cold shock proteins and virulence factors. Furthermore, 125 genes were up-regulated in the presence of lactoferrin, comprising genes involved in sporulation and germination, nutrient uptake, iron transport and utilization, and resistance. In summary, lactoferrin exposure of B. cereus strain-specifically triggers an extensive transcriptional response that considerably exceeds the response toward iron deficiency and, despite down-regulation of various genes belonging to the PlcR-regulon, ultimately leads to an increased level of secreted enterotoxin by a mechanism, which has yet to be elucidated

Consumed Foodstuffs Have a Crucial Impact on the Toxic Activity of Enteropathogenic Bacillus cereus

Enteropathogenic Bacillus cereus cause diarrhea due to the production of enterotoxins in the intestine. To start this process, spores have to be ingested together with contaminated food and survive the stomach passage. In this study, the influence of consumed foodstuffs on spore survival as well as on cytotoxicity toward colon epithelial cells was investigated. Spore survival of 20 enteropathogenic and apathogenic B. cereus strains during simulated stomach passage was highly strain-specific and did not correlate with the toxic potential. Survival of three tested strains was strain-specifically altered by milk products. Whereas milk, a follow-on formula and rice pudding had only little influence, spores seemed to be protected by milk products with high fat content such as whipped cream and mascarpone. Furthermore, tested milk products decreased the toxic activity of three B. cereus strains toward CaCo-2 cells. Investigating the individual components, lactoferrin, a skim milk powder and vitamins C, B5 and A showed the most inhibiting effects. On the other hand, biotin, vitamin B3 and another skim milk powder even enhanced cytotoxicity. Further studies suggested that these inhibiting effects result only partially from inhibiting cell binding, but rather from blocking the interaction between the single enterotoxin components

From genome to toxicity: a combinatory approach highlights the complexity of enterotoxin production in Bacillus cereus

In recent years Bacillus cereus has gained increasing importance as a food poisoning pathogen. It is the eponymous member of the Bacillus cereus sensu lato group that consists of eight closely related species showing impressive diversity of their pathogenicity. The high variability of cytotoxicity and the complex regulatory network of enterotoxin expression have complicated efforts to predict the toxic potential of new Bacillus cereus isolates. In this study, comprehensive analyses of enterotoxin gene sequences, transcription, toxin secretion and cytotoxicity were performed. For the first time, these parameters were compared in a whole set of Bacillus cereus strains representing isolates of different origin (food or food poisoning outbreaks) and of different toxic potential (enteropathogenic and apathogenic) to elucidate potential starting points of strain-specific differential toxicity. While toxin gene sequences were highly conserved and did not allow for differentiation between high and low toxicity strains, comparison of nheB and hblD enterotoxin gene transcription and Nhe and Hbl protein titers revealed not only strain-specific differences but also incongruence between toxin gene transcripts and toxin protein levels. With one exception all strains showed comparable capability of protein secretion and so far, no secretion patterns specific for high and low toxicity strains were identified. These results indicate that enterotoxin expression is more complex than expected, possibly involving the orchestrated interplay of different transcriptional regulator proteins, as well as posttranscriptional and posttranslational regulatory mechanisms plus additional influences of environmental conditions

Bacillus cereus non-haemolytic enterotoxin activates the NLRP3 inflammasome

Inflammasomes are important for host defence against pathogens and homeostasis with commensal microbes. Here, we show non-haemolytic enterotoxin (NHE) from the neglected human foodborne pathogen Bacillus cereus is an activator of the NLRP3 inflammasome and pyroptosis. NHE is a non-redundant toxin to haemolysin BL (HBL) despite having a similar mechanism of action. Via a putative transmembrane region, subunit C of NHE initiates binding to the plasma membrane, leading to the recruitment of subunit B and subunit A, thus forming a tripartite lytic pore that is permissive to efflux of potassium. NHE mediates killing of cells from multiple lineages and hosts, highlighting a versatile functional repertoire in different host species. These data indicate that NHE and HBL operate synergistically to induce inflammation and show that multiple virulence factors from the same pathogen with conserved function and mechanism of action can be exploited for sensing by a single inflammasome

Binding to The Target Cell Surface Is The Crucial Step in Pore Formation of Hemolysin BL from Bacillus cereus

A major virulence factor involved in Bacillus cereus food poisoning is the three-component enterotoxin hemolysin BL. It consists of the binding component B and the two lytic components L1 and L2. Studying its mode of action has been challenging, as natural culture supernatants additionally contain Nhe, the second three-component enterotoxin, and purification of recombinant (r) Hbl components has been difficult. In this study, we report on pore-forming, cytotoxic, cell binding and hemolytic activity of recently generated rHbl components expressed in E. coli. It is known that all three Hbl components are necessary for cytotoxicity and pore formation. Here we show that an excess of rHbl B enhances, while an excess of rHbl L1 hinders, the velocity of pore formation. Most rapid pore formation was observed with ratios L2:L1:B = 1:1:10 and 10:1:10. It was further verified that Hbl activity is due to sequential binding of the components B - L1 - L2. Accordingly, all bioassays proved that binding of Hbl B to the cell surface is the crucial step for pore formation and cytotoxic activity. Binding of Hbl B took place within minutes, while apposition of the following L1 and L2 occurred immediately. Further on, applying toxin components simultaneously, it seemed that Hbl L1 enhanced binding of B to the target cell surface. Overall, these data contribute significantly to the elucidation of the mode of action of Hbl, and suggest that its mechanism of pore formation differs substantially from that of Nhe, although both enterotoxin complexes are sequentially highly related

The Food Poisoning Toxins of Bacillus cereus

Bacillus cereus is a ubiquitous soil bacterium responsible for two types of food-associated gastrointestinal diseases. While the emetic type, a food intoxication, manifests in nausea and vomiting, food infections with enteropathogenic strains cause diarrhea and abdominal pain. Causative toxins are the cyclic dodecadepsipeptide cereulide, and the proteinaceous enterotoxins hemolysin BL (Hbl), nonhemolytic enterotoxin (Nhe) and cytotoxin K (CytK), respectively. This review covers the current knowledge on distribution and genetic organization of the toxin genes, as well as mechanisms of enterotoxin gene regulation and toxin secretion. In this context, the exceptionally high variability of toxin production between single strains is highlighted. In addition, the mode of action of the pore-forming enterotoxins and their effect on target cells is described in detail. The main focus of this review are the two tripartite enterotoxin complexes Hbl and Nhe, but the latest findings on cereulide and CytK are also presented, as well as methods for toxin detection, and the contribution of further putative virulence factors to the diarrheal disease.publishedVersio

Characteristics of the Protein Complexes and Pores Formed by Bacillus cereus Hemolysin BL

Bacillus cereus Hemolysin BL is a tripartite toxin responsible for a diarrheal type of food poisoning. Open questions remain regarding its mode of action, including the extent to which complex formation prior to cell binding contributes to pore-forming activity, how these complexes are composed, and the properties of the pores formed in the target cell membrane. Distinct complexes of up to 600 kDa were found on native gels, whose structure and size were primarily defined by Hbl B. Hbl L1 and L2 were also identified in these complexes using Western blotting and an LC-MS approach. LC-MS also revealed that many other proteins secreted by B. cereus exist in complexes. Further, a decrease of toxic activity at temperatures ≥60 °C was shown, which was unexpectedly restored at higher temperatures. This could be attributed to a release of Hbl B monomers from tight complexation, resulting in enhanced cell binding. In contrast, Hbl L1 was rather susceptible to heat, while heat treatment of Hbl L2 seemed not to be crucial. Furthermore, Hbl-induced pores had a rather small single-channel conductance of around 200 pS and a probable channel diameter of at least 1 nm on planar lipid bilayers. These were highly instable and had a limited lifetime, and were also slightly cation-selective. Altogether, this study provides astonishing new insights into the complex mechanism of Hbl pore formation, as well as the properties of the pores

The Food Poisoning Toxins of Bacillus cereus

Bacillus cereus is a ubiquitous soil bacterium responsible for two types of food-associated gastrointestinal diseases. While the emetic type, a food intoxication, manifests in nausea and vomiting, food infections with enteropathogenic strains cause diarrhea and abdominal pain. Causative toxins are the cyclic dodecadepsipeptide cereulide, and the proteinaceous enterotoxins hemolysin BL (Hbl), nonhemolytic enterotoxin (Nhe) and cytotoxin K (CytK), respectively. This review covers the current knowledge on distribution and genetic organization of the toxin genes, as well as mechanisms of enterotoxin gene regulation and toxin secretion. In this context, the exceptionally high variability of toxin production between single strains is highlighted. In addition, the mode of action of the pore-forming enterotoxins and their effect on target cells is described in detail. The main focus of this review are the two tripartite enterotoxin complexes Hbl and Nhe, but the latest findings on cereulide and CytK are also presented, as well as methods for toxin detection, and the contribution of further putative virulence factors to the diarrheal disease

Binding to The Target Cell Surface Is The Crucial Step in Pore Formation of Hemolysin BL from <i>Bacillus cereus</i>

A major virulence factor involved in Bacillus cereus food poisoning is the three-component enterotoxin hemolysin BL. It consists of the binding component B and the two lytic components L1 and L2. Studying its mode of action has been challenging, as natural culture supernatants additionally contain Nhe, the second three-component enterotoxin, and purification of recombinant (r) Hbl components has been difficult. In this study, we report on pore-forming, cytotoxic, cell binding and hemolytic activity of recently generated rHbl components expressed in E. coli. It is known that all three Hbl components are necessary for cytotoxicity and pore formation. Here we show that an excess of rHbl B enhances, while an excess of rHbl L1 hinders, the velocity of pore formation. Most rapid pore formation was observed with ratios L2:L1:B = 1:1:10 and 10:1:10. It was further verified that Hbl activity is due to sequential binding of the components B - L1 - L2. Accordingly, all bioassays proved that binding of Hbl B to the cell surface is the crucial step for pore formation and cytotoxic activity. Binding of Hbl B took place within minutes, while apposition of the following L1 and L2 occurred immediately. Further on, applying toxin components simultaneously, it seemed that Hbl L1 enhanced binding of B to the target cell surface. Overall, these data contribute significantly to the elucidation of the mode of action of Hbl, and suggest that its mechanism of pore formation differs substantially from that of Nhe, although both enterotoxin complexes are sequentially highly related